This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To study a new vaccine approach to HIV. Immune epitope dominance results in one or a few epitopes comprising the greatest part of the immune response to SIV infection and vaccination. We know that some of these dominant epitopes are not useful in controlling SIV because they either mutate rapidly (Mamu-A*01 Tat SL8) or simply do not control viremia on their own (Mamu-A*01 Gag CM9). It may be that subdominant immune responses are more efficacious than dominant immune responses, but are at low frequency. Therefore, we have designed a series of Minigenes, which are regions of SIV proteins that encode no more than one immunodominant epitope. By separating immunodominant epitopes from subdominant epitopes, we hope to allow increased frequency of the subdominant epitopes resulting in greater breadth of the immune response. In addition, while we have had successful vaccine trials in rhesus macaques with DNA/Ad5 as vaccine vectors, adenovirus is now less favored as a vaccine vector in humans due to widespread prior infection with adenovirus and due to the poor results obtained in the recent Merck STEP trial using this vector. Therefore, at the same time that we are exploring the minigene concept, we are also exploring the use of alternate vectors. We will put minigenes into rBCG, rShigella, rYF-17D and possibly other vectors, vaccinate macaques and challenge them to assess the immunogenicity of these new approaches. PROGRESS: Following up on our previous publication (see Novel, Logical grant) we have started a new vaccine/challenge study with the goal of enhancing breadth of Gag, Vif and Nef, as well as to test new vectors and delivery systems. We are just finished with the vaccinations and will begin challenging in February 2011. In this study, we inserted six minigenes of Gag, two minigenes of Vif and the core region of Nef into DNA plasmids, recombinant YF-17D vectors and Ad5 vectors. These minigenes were optimized for expression by our colleagues at IAVI. We utilized several different vaccine strategies to compare use if IL-12 during vaccination, use of rYF as either a boost or a prime and comparison of DNA prime/Ad5 boost to rYF prime/Ad5 boost. For the DNA primes, we used a new technique called DNA electroporation, which greatly enhanced immune responses to the sequences encoded in the DNA plasmids. In one group, these responses were further enhanced by the addition of a plasmid encoding IL-12. We found that these immune responses were greatly biased toward CD4+ T cell responses. Subsequent rYF and/or rAd5 boosts resulted in a more balanced immune response, with the result that we have robust immune responses in both CD4+ and CD8+ T cells compartments. We also primed with rYF, then boosted with rAd5, because colleagues have shown that rYF acts more like a prime than a boost. Comparison of the responses engendered by this regimen confirmed that while the responses observed after rYF prime are low, the responses seen after boosting with rAd5 are much greater than that observed with rAd5 alone. Now that these vaccinations are completed, we will start challenging animals in February 2011. This research used WNPRC Animal Services, Genetics Services, and Immunology &Virology Services. This grant also utilized the resources of the MHC typing facility and Immunology and Virology Services (viral load determination, tetramers, flow cytometry instrument utilization, Elispots). PUBLICATION: Sacha JB, Buechler MB, Newman LP, Reed J, Wallace LT, Loffredo JT, Wilson NA, Watkins DI. Simian immunodeficiency virus-specific CD8+ T cells recognize Vpr- and Rev-derived epitopes early after infection. J Virol. 2010 Oct;84(20):10907-12. Epub 2010 Aug 4. PubMed PMID: 20686015;PubMed Central. PMCID: PMC2950557.